The Section on Directed Gene Delivery assesses the ability of retroviral vectors to deliver genes to the central nervous system and the role of retroviruses in various neurological disorders. Over the course of last year the Section has made improvements to the design of viral vectors used for therapeutic gene delivery to patients and has identified factors critical to infection of human cells by both therapeutic viral vectors based on retroviruses and the wild type retroviral pathogens themselves. It has been a widely held belief that gammaretroviral vectors, in contrast to lentiviral vectors, are restricted in their ability to transduce growth-arrested cells, yet the block to this restriction has not been clearly defined. We have compiled findings regarding the ability of gammaretoviral vectors and replicating gammaretroviruses that express green fluorescent protein as an indicator of infection to infect non-dividing target cells. We are also in the process of examining the defects in self-inactivating (SIN) retroviral vectors currently employed in therapeutic gene delivery. SIN vectors are based on the premise that vectors in which the viral promoters are removed will not activate expression of adjacent genes upon integration into the target cells'genome and thus eliminating the genotoxicity associated with non-SIN vectors. We have discovered the presence of a strong cryptic promoter present in commercially available SIN retroviral vectors. We have mapped this region and have excised it without compromising the performance of vectors. Human gene therapy often relies on retrovirus- or lentivirus-based platforms for stable integration of transgenes into the genome of host cells. However, it has long been noticed that retroviruses are subject to silencing in embryonic stem cells. Replicators are DNA sequences that initiate early DNA replication, block transcriptional silencing and maintain DNA acetylation patterns characteristic of euchromatin. Inclusion of functional replicators in gene therapy vectors may rescue transcriptional silencing of the therapeutic gene when integrated into certain regions of the genome. This possibility is being explored in collaboration with Mirit Aladjem at the NCI who originally characterized replicator sequences. The second focus of our section's research is based on determining whether gammaretroviral agents are etiologically liked to neurological disorders in humans. It has been reported that gammaretroviruses are present in the US population at a frequency that exceeds domestic HIV-1 infection rates. The gammaretrovirus XMRV has been isolated from patients with prostate cancer and more recently with chronic fatigue syndrome (CFS) and in children diagnosed with neuroimmune disorders. The NIH has sponsored CFS research for many years and is committed to increasing both the amount and quality of research in this area. Through September 1999, the National Institute of Allergy and Infectious Diseases (NIAID) held sole responsibility for research in this area. In October 1999, Dr. Harold Varmus, in consultation with Dr. Anthony Fauci, recognizing that a multidisciplinary and integrated approach encompassing the missions of many NIH ICs was necessary to address CFS, incorporated responsibility into the Office of the Director (OD). Our lab is recognized for our research on a similar gammaretrovirus isolated from nonhuman primates, gibbon ape leukemia virus (GALV). GALV is the only gammaretrovirus other than XMRV found in primates. In collaboration with Frank Ruscetti at the NCI, Bill Switzer at the CDC and Suzan Winfield and Jessica Siegal-Willcot at the National Zoo, we are investigating the source animal for XMRV, and screening gibbon apes in US zoos for the presence of GALV and XMRV. We have obtained samples from the CDC and the Biological Research Steering Committee that provide us with materials permitting us to determine that many gibbon apes at various zoos have been infected with an XMRV-like virus. We have determined the cell tropism of XMRV using an engineered biologically active XMRV virus with a GFP reporter gene and are identifying cellular factors that restrict XMRV infection of receptor bearing cells. These factors that can restrict XMRV infection will be used as a means of developing XMRV antiviral drugs. In addition to being a horizontally transmitted infectious agent, XMRV is a threat to the human genome. We are in the process of isolating rat germ line cells and exposing these cells to engineered biologically active XMRV virus with a GFP reporter. Sperm obtained from these cells are being assessed. Positive results of sperm expressing GFP indicate that XMRV can be transmitted from infected individuals horizontally (i.e., to offspring as a mendelian trait) as well as vertically through the more traditionally route of viral infection. We used cysteine scanning mutagenesis (SCAM) methods to assess the topology of the GALV receptor and intend to identify extracellular domains of the XMRV receptor using similar methods. These studies will lead to the identification of the XMRV-binding domain. Finally the spread of most retroviruses is mediated not by direct virus infection but by cell-cell transmission from an infected cell to an uninfected cell. We have developed a model system to assess blocks to cell-cell virus transmission using spinning-disc confocal microscopy to visualized individual budding of fluorescently labeled virus particles into adjacent cells in three dimensional space over time.